Botanical Studies (2011) 52: 265-275.

BIOCHEMISTRY

Increased cellulose production by heterologous
expression of cellulose synthase genes in a filamentous
heterocystous cyanobacterium with a modification in
photosynthesis performance and growth ability

Hsiang-Yen SU^1,4, Tse-Min LEE²'³'⁴, Yu-Lu HUANG^2,3, Sheng-Hsin CHOU⁵, Jia-Baau WANG⁵, Li-
Fu LIN⁵, and Te-Jin CHOW¹*

¹Department of Biotechnology, Fooyin University, 151, Chinhsueh Rd., Ta-liao, Kaohsiung 831, Taiwan
²Institute of Marine Biology, National Sun Yat-sen Uni^ersit^, Kaohsiung 804, Taiwan

³The Kuroshio Research Group of the Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan

⁴Doctor Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 804, Taiwan
⁵Institute of Nuclear Energy Research, Atomic Energy Council, Executive Yuan, Taoyuan County 325, Taiwan

(Received May 26, 2010; Accepted October 8, 2010)

ABSTRACT. Cellulose and sugar from microalgae can be utilized for the production of biofuel ethanol. Increasing cellulose and sugar synthesis capacity is key for high yield production of this biofuel. To enhance cellulose production, we transferred acsAB, responsible for cellulose synthesis in bacterium Acetobacter xy-linum, into the cyanobacterium, Anabaena sp. strain PCC7120, by conjugation. PCR confirmed the presence of acsAB in Anabaena sp. strain PCC7120 exconjugates. RT-PCR demonstrated the up-regulation of acsAB expression. Production of extracellular cellulose secreted from Anabaena sp. strain PCC7120 carrying acsAB was revealed using Calcofluor white staining and cellobiohydrolase I (CBHI)-FITC labeling. Further evidence obtained from the digestion of cellulose to glucose demonstrated that the amount of glucose released from cellulose was significantly increased in Anabaena sp. PCC7120 cells carrying acsAB as compared to the wild type. The photosynthetic efficiency and growth rate were increased in the transgenic strains. Cellulose synthesis is thus enhanced in Anabaena sp. strain PCC7120 expressing acsAB and does not negatively impact photosynthesis and growth.

Keywords: Cellulose; Cellulose synthase; Cyanobacteria; Photosynthesis.

INTRODUCTION

Patzek, 2008). Additionally, the cultivation of cellulose crops, e.g. cotton for textiles or switchgrass for biofuels, entails the extensive use of arable land (Sun and Cheng, 2002), fertilizers and pesticides, as well as the consumption of fresh water for irrigation (Pimentel and Patzek, 2008). Considering Taiwan's rich and vast microalgal resources, microalgal-derived cellulose stands as a promising alternative to traditional plant cellulose sources.

Cyanobacteria, also known as blue-green algae, are a group of photoautotrophic prokaryotes that perform plantlike oxygenic photosynthesis. These organisms have simple growth requirements and efficiently use light, CO2, and inorganic elements to produce biomass. Many of them are non-toxic, have high nutrient value, and can be easily cultivated for mass commercial production (Kay and Barton, 1991). They are attractive systems for the bioconversion of solar energy and CO₂ into valuable products. The genetic characteristics of cyanobacteria are similar to E. coli, in that their genetic manipulation is relatively easy. This has

Renewable energy sources, notably ethanol from bio-mass, are currently being developed as eventual replacements for present non-renewable energy sources such as petroleum. The limiting factors for producing ethanol from biomass include their high cost and the limited availability of biomass sources, such as corn, sugar cane and cellulose from plants (Sanchez and Cardona, 2008; Balat and Balat, 2009). The cellulose of plant cell walls is intimately associated with lignin and hemicelluloses, forming a complex polymer composite that is exceptionally recalcitrant to mechanical and biological degradation (Sun and Cheng, 2002; Lynd, 1996). Cellulose purification is both energetically expensive and environmentally unfriendly (Pimentel and

*Corresponding author: E-mail: dr008@mail.fy.edu.tw; Tel: 886-7-7811151 ext. 7907; Fax: 886-7-7862707.

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made them model systems in molecular biological studies. The fact that most cyanobacterial strains produce no endotoxin makes them attractive hosts for the production of recombinant products since the purification process is easier and the production less costly.

In recent years, rapid advancements have been made in the genetic engineering of cyanobacteria. The physiology, biochemistry and genetics of Anabaena sp. PCC7120, a filamentous heterocystous cyanobacterium, have been well characterized. Anabaena sp. PCC7120 has a well-developed genetic transfer system that has been used to express a number of foreign genes in this organism. For example, expression of the mouse metallothionein-I gene in transgenic Anabaena sp. PCC7120 conferred cadmium resistance (Ren et al., 1998), while combinational expression of several toxin genes from Bacillus thuringiensis conferred mosquito larvicidal activity to transgenic Ana-baena sp. PCC 7120 (Khasdan et al., 2003; Khasdan et al., 2003; Manasherob et al., 2002; Wu et al., 1997). The human tumor necrosis factor alpha (hTNF-a) gene was also expressed in Anabaena sp. PCC 7120 resulting in cytotox-icity of the crude extracts from the transgenic cyanobacte-ria (Liu et al., 1999; Dai et al., 2001). ；

Acetobacter xylinum is a gram-negative bacterium which produces a cellulose membrane called bacterial cellulose (BC) (Marx-Figini and Pion, 1974). This highly-pure and mechanically-strong membrane can be used as a food additive and in medical materials (Czaja et al., 2006). The acsABCD operon encodes the enzymes responsible for cellulose biosynthesis in Acetobacter xylinus (Glucon-acetobacter xylinus). The cellulose synthases of A. xylinum ATCC23769 and ATCC53582 contain three subunits (Acs-AB, AcsC, and AcsD). The deduced amino acid sequences of the cellulose synthases of A. xylinum ATCC23769 and ATCC53582 are highly homologous (Saxena et al., 1994; Kawano et al., 2002). The N-terminal half of AcsAB catalyzes β-glycosyltransfer using either UDP-glucose or similar nucleotide sugars as a substrate (Saxena et al., 1995). The C-terminal half of AcsAB is involved in the regulation of cellulose biosynthesis, as it interacts with cyclic-di-GMP (Ross et al., 1987), an activator of cellulose production. Although the precise functions of AcsC and AcsD have not yet been determined, it has been proposed that AcsC might form pores in the cell wall through which newly-synthesized cellulose can pass, while AcsD might influence cellulose crystallization (Saxena et al., 1994).

Recently, Nobles and Brown (2008) reported the transfer of a partial cellulose synthesis operon containing acs-ABDC, from the Gluconacetobacter xylinus strain ATCC 53582 to the unicellular cyanobacterium Synechococcus leopoliensis strain UTCC 100, resulting in the secretion of non-crystalline cellulose and sugar. In the current study, a shuttle expression vector was constructed, harboring the cellulose synthethase gene, acsAB, from Acetobacter xylilum ATCC23769, under the control of the psbA promoter, and introduced by conjugation into the filamentous, heterocystous cyanobacterium Anabaena sp. PCC

7120, The production of cellulose, as a result of acsAB expression, was detected by Calcofluor white staining and FITC-labelled CBHI. Given the possibility that enhanced cellulose synthesis might inhibit the growth of acsAB transgenic Anabaena the growth and photosynthetic capacity, as measured by cell density and chlorophyll a content, respectively, were compared between the wild type and acsAB transgenic Anabaena.

MATERIALS AND METHODS

Plasmids and bacterial strains

The bacterial strains and plasmids used are listed in Table 1. A. xylinum ATCC23769 was grown in mannitol agar containing: 2.5% mannitol, 0.5% yeast extract, 0.3% peptone and 1.5% agar (ATCC MEDIA #1 Agar: Mannitol Agar) at 28°C. Escherichia coli cells were grown in LB medium (Miller, 1972) at 37°C on a rotary shaker. Selection with kanamycin (Km) was done at a concentration of 50 fig/mL. All recombinant plasmid constructions used E. coli DH5a as a host. Professor C. P. Wolk of Michigan State University (USA) kindly supplied Plasmid pRL489. Anabaena sp. PCC 7120 was obtained from the Pasteur Institute (Paris, France).

Growth conditions

Anabaena sp. strain PCC 7120 was cultured in 50 mL modified BG11 medium (BG-11 supplemented with 2 x Na2C〇3) in a 125 mL flask containing 15 fig/mL neomycin. Algal cells, with initial OD750 of 0.1, were incubated in a rotary shaker (TKS OSI-500R, Kaohsiung, Taiwan) at 28°C at a speed of 130 rpm with continuous irradiance with 50 fimol photons m^-2 s^-1. After 15 days of incubation, algal cells were collected and the OD750 and chlorophyll a content were measured. Photosynthesis and respiration rates were also determined. Transgenic strains grown on plates were maintained on solid agar media containing neomycin at 25 fg/mL.

Cloning acsAB genes from A. xylinum

Total genomic DNA from A. xylinum ATCC23769 was isolated and used as a template to amplify the acsAB genes by polymerase chain reaction (PCR) using forward (acs-AB-F: 5'-aattggatccatgccagaggttcggtcgtcaacgcagtca-3') and reverse (acsAB-R: 5'-aattggatcctcacgacttgcgcctct-catcctcaagctg-3') primers. The initiation and termination codons of acsAB were underlined in the primers. PCR was carried out in a volume of 50 fL with the following parameters: 95°C for 3 min; 30 cycles at 98°C for 20 sec and 68°C for 7 min: and a final extension at 72°C for 10 min using Takara LA Taq polymerase (Takara Shuzo, Kyoto, Japan). The PCR product with the expected size (4.6kb) was isolated and cloned into yT&A vector (Yeast-ern Biotech Co., Ltd, Taipei, Taiwan) resulting in plasmid pAcsAB. Complete nucleotide sequence analysis of the acsAB insert in pAcsAB was confirmed by DNA sequencing (Tri-I Biotech, Taipei, Taiwan).

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Table 1. Bacterial strains and plasmids used.
Strain or plasmid	Relevant characteristic (s)	Source or reference
Strains of Anabaena sp.
PCC 7120	Wild type	PCC^a
Strain acsAB-2	PCC 7120 carrying pPacsAB	This study
Strain acsAB-8	PCC 7120 carrying pPacsAB	This study
Strain acsAB-17	PCC 7120 carrying pPacsAB	This study
Strain acsAB-26	PCC 7120 carrying pPacsAB	This study
Strain pRL489-Nm^r	PCC 7120 carrying pRL489-Nm^r	This study
Strains of E. coli
DH5a	Recipient in transformations
Strains of A. xylinum
A. xylinum ATCC23769	Source of acsAB gene	ATCC^b
Plasmids
pAcsAB	yT&A containing 4.6 kb acsAB gene	This study
pRL489-Nm^r	pRL489 removing luxAB gene	This study
pPacsAB	pRL489-Nm^r containing acsAB gene	This study
pRL489	Km^r Nm^r; pDU1-based shuttle vector; carries PpsbA-luxAB	Elhai, J.
pRL443	Ap^r Tc^r; conjugative plasmid	Elhai, J. & C. P. Wolk
pRL528	Cm^r; helper plasmid; carries mob	Elhai, J. & C. P. Wolk
yT&A	Ap^r; cloning vector	Yeastern Biotech Co.

^aPCC, The Pasteur Culture Collection of Cyanobacteria, Institut Pasteur, France.

^bATCC, American Type Culture Collection, Rockville, MD, USA.

Triparental conjugative transfer

Conjugation between E. coli and Anabaena was performed as described (Elhai and Wolk, 1988). Briefly, two strains of E. coli HB101: one containing conjugation plasmid, pRL443, and the other containing helper plas-mid, pRL528, along with either shuttle vector pPacsAB, or pRL489-Nm^r (control), were mixed with Anabaena sp. PCC 7120 and spotted onto filter papers laid on BG-11 agar plates without antibiotic. After incubation for 48 h, the filters were transferred to plates supplemented with 25 [ig/mL neomycin (Nm) and incubated until Nm^r exconju-gant colonies appeared. The cyanobacterial clones, which appeared on the filters after about two weeks, were picked and restreaked on BG-11 plates containing 25 [ig/mL neo-mycin (Nm). Putative exconjugates were transferred to BG-11 liquid medium containing Nm (15 [g/mL) for more rapid cultivation and further analysis.

Shuttle vector construction

A 2 kb BamHI fragment containing the luxAB sequence was removed from pRL489 (Elhai, 1993) and a 4.6 kb BamHI fragment from pAcsAB, containing the acsAB genes from A. xylinus strain ATCC 23769, was ligated into the BamHI site of pRL489 to create a shuttle plasmid pPacsAB, where the acsAB sequence was oriented in the same direction as the psbA promoter (Figure 1).

The 2 kb BamHI fragment containing the luxAB sequence was removed from pRL489 and the remaining fragment was self-ligated to generate plasmid pRL489-Nm^r. Plasmid pRL489-Nm^r was used as a negative control in detecting acsAB expression in acsAB transgenic cells. The resulting plasmids, pRL489-Nm^r and pPacsAB, were confirmed by restriction digestion, and used for conjuga-tive transfers.

PCR screens for transgenic cells

Cultures of Anabaena sp. PCC7120 harboring either plasmid pRL489-Nm^r or pPacsAB were used in PCR screens to detect acsAB. One mL liquid culture of each strain was pelleted by centrifugation, resuspended in 200 fiL of TE, pH 8.0 supplemented with 1% Triton X-100, and lysed by heating for 2 min at 95°C (Hagen and Meeks, 1999). The lysate was then extracted twice with an equal

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volume of chloroform. Aliquots (5 iL) of the aqueous phase, which contained genomic DNA extracted from Ana-baena sp. PCC7120 wild type and Ananbaena PCC7120 transformed with pRL489-Nm^r or pPacsAB were used in PCR amplifications with acsAB-F and acsA-R primers.

PCR was carried out in a Thermo Hybaid Px2 thermal cycler (Thermo, Franklin, MA, USA) using the following temperature program: 95°C for 5 min; 30 cycles consisting of 95°C for 1 min, 52°C for 1 min, and 72°C for 2 min; and 72°C for 10 min. A 2.2 Kb fragment spanning the acsA region of the acsAB genes was amplified using the primers acsAB-F 5'-aattggatccatgccagaggttcggtcgtcaacgcagtca-3' and acsA-R 5' -aattcccgggtcagacagggcattgccagccgaaggct-tg-3' (the underlined regions of the primer sequences indicate introduced restriction endonuclease sites). The resulting PCR products were then detected by agarose gel electrophoresis.

Conjugation of Cellobiohydrolase I with fluorescent probes

The conjugation of Cellobiohydrolase I (CBH I) to fluorescein isothiocyanate (FITC) was carried out according to the manufacturer's instructions (Pierce® FITC Antibody Labeling Kit, Thermo Scientific No. 53027, Rockford, USA). One mg CBH I (Cellobiohydrolase I from Trichoderma sp., Megazyme Lot 40203a, Sidney, Australia) was mixed with FITC in 0.05 M borate buffer (pH 8.5). After incubation at room temperature for 1 hr in the dark, the FITC labeled Cellobiohydrolase I (FITC-CBHI) was separated from unconjugated dye by chroma-tography on spin columns.

Specimen fixation and cellulose labeling with FITC-conjugated CBHI

One mL of liquid cultures of Anabaena PCC7120 wild type and Anabaena PCC7120 harboring plasmid pRL489-Nm^r, pPacsAB were pelleted by centrifugation, and resuspended in 200 [l of ethanol/acetic acid solution (ethanol:acetic acid = 3:1) at room temperature for 1 hr. After centrifugation, the cell pellets were resuspended in 50 [L of 50 mM sodium acetate buffer (pH 5.0). 20 [L of the suspension was spotted onto a poly-L-lysine coated slide. The slides were immersed in CBHI-FITC (1:20 diluted with PBS) and incubated at room temperature for 30 min. They were then rinsed four times for five minutes with PBS. The samples were mounted to a coverslip with ibidi mounting medium (Ibidi, Martinsried, Germany).

After staining, fluorescence and phase contrast micrographs of the CBHI-FITC labeled Anabaena samples were taken on a LEICA microscope (LEICA DM2500) with a 25X and a 40X objective using specific emission filter sets (Ex.450-490 nm, Em. 510-550 nm). Images were cap-tured with a CoolSnap ES CCD camera (Roper Scientific, Tucson, AZ, USA) attached to the microscope.

RNA isolation and RT-PCR

Total RNA was isolated from cultures of Anabaena

strains using the GENEMARK Plant Total RNA Miniprep Purification Kit (Taipei, Taiwan). The RNA quality was analyzed by agarose gel electrophoresis (1%) and the con-centration was determined by O.D. at 260 nm (T60 UV-Visible spectrophotometer, PG Instruments, Leicestershire, United Kingdom).

Reverse transcription reactions were carried out with M-MLV Reverse Transcriptase (catalog # M1701, Pro-mega, Madison, Wisconsin, USA) based on the manufacturer's instructions, using random primers and 1[ig of DNAse-treated RNA. cDNA produced in reverse transcription reactions was used for PCR reactions with acsAB-F and acsA-R primers. The following PCR program profile was applied: 1 min at 94°C followed by 40 cycles of time and temperature; 10 s denaturation at 94°C, 1 min annealing at 55°C, and 2 min elongation at 72°C; finishing with 7 min at 72°C. Products of the PCR reactions were analyzed on 1% agarose gels. Negative controls in which no reverse transcriptase was added to the RT reaction prior to PCR and reactions in which dH₂O was added to the PCR in place of DNA were included in the experiments.

Cellulose hydrolysis and determination of glucose contents

Cells from 15 mL of stationary phase cultures of Ana-baena PCC7120 wild type, and Anabaena PCC7120 cells harboring plasmid pRL489-Nm^r, pPacsAB were collected by centrifugation at 3,000 x g for 10 min. The pellets were resuspended in 0.6 mL of sodium acetate buffer (50 mM sodium acetate, pH 5.0) and broken by shaking for 1 minute using a minibead beater (Biospec Products, Bartlesville, OK, USA) in the presence of 0.2 g sea sand. For total cellulose hydrolysis analysis, 24 [L of sterilized Celluclast 1.5 L and 4 [iL of 188 (Novozymes) were added to the broken cells, followed by incubation at 50^oC for 48 h. The low-molecular-mass compounds were extracted with 1 mL of 80% (v/v) ethanol for 3 h at 65oC. After cen-trifugation at 14,000 rpm for 2 min at room temperature, the supernatant was collected and the pellet was washed again with 0.5 mL of 80% ethanol and centrifuged. Both supernatants were combined and incubated for 30 min at 80oC (Higo et al., 2006). The amount of glucose in these

Calcofluor white staining and microscopy

In order to detect extracellular cellulose, Calcofluor white M2R (Sigma F3543) was used to stain the transgen-ic strains of Anabaena PCC7120. An aqueous solution of Calcofluor white M2R (Sigma F3543) of 0.01% (w/v) was overlaid on cells fixed to microscope slides and incubated for about 5 min at room temperature. After staining, fluo-rescence and phase contrast micrographs were taken on a LEICA microscope (LEICA DM2500) with an X100 ob-jective using specific emission filter sets (Ex. 377 nm, Em. 447 nm). Images were captured with a cool CCD camera (Leica model DFC420C, Wetzlar, Germany).

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samples was determined with a modified glucose oxidase-peroxidase coupled reaction as previously described (Ou-Lee and Setter, 1985; Cheng, 1994). In brief, PGO reagent (50 mM HEPES containing 3 mg/mL p-hydroxybenzoic acid, 0.1 mg/mL 4-aminoantipyrrine, 0.5 units peroxidase, and 1.5 units glucose oxidase, pH 7.0) was added to the ethanol-extracted supernatant, followed by incubation at room temperature for 15 min. The absorbance at 490 nm was obtained using a Microplate Spectrophotometer (iQuant, Bio-Tek Instruments, Inc. USA). A series of glucose dilutions was included in the assay as a standard.

RESULTS AND DISCUSSION

Construction of expression shuttle vectors of Anabaena sp. PCC7120

For cellulose production in Anabaena sp. strain PCC 7120, the shuttle expression plasmid pPacsAB was constructed using a pDU1 derivative shuttle vector pRL489 (Elhai and Wolk, 1988). The acsAB-containg DNA frag-ment of A. xylinum was amplified by PCR and cloned into the yT&A vector to generate plasmid pAcsAB. DNA sequencing confirmed the cloned acsAB sequence which was compared with other known proteins using the NCBI BLAST algorithm (http://blast.ncbi.nlm.nih.gov). As shown in Figure 1, the expression of the acsAB genes in plasmid pPacsAB was under the control of the strong psbA promoter derived from the chloroplast DNA of Amaran-thus hybris (Hirschberg and McIntosh, 1983).

Determination of algal cell growth rate and chlorophyll a contents

Algal cell growth was estimated by OD₇₅₀ in 1 mL of cell suspension using spectrophotometer (HITACHI U-2001, Japan).

To determine the chlorophyll a content, 10 mL of algal cell culture was centrifuged at 12,000 x g at 4^oC for 5 min, the pellet was ground in liquid nitrogen, extracted with 10 mL of chilled 80% acetone at 4^oC and shaken in the dark at 100 rpm for 10 minutes. After centrifugation at 12,000 xg at 4^oC for 5 min, the supernatant was collected and the volume was adjusted to 10 mL with 80% acetone. The absorbance of the supernatant was detected at 645 nm and 663 nm for the estimation of chlorophyll a contents. The content of chlorophyll a was determined using the equation: Chl a (μg/mL) = 12.7(A₆₆₃)- 2.69 (A₆₄₅)(Arnon,

1949).

Conjugative transfer and the selection of the transgenic cyanobacterial cells

The shuttle expression plasmid pPacsAB was introduced to Anabaena cells by triparental conjugaton, and exconjugants were selected on BG-11 agar plates containing neomycin (25 [g/ml). Plasmid pRL489-Nm^r was introduced to Anabaena cells by triparental conjugation and a resulting exconjugant was used as a negative control in cellulose production assays. Transgenic cyanobacterial clones forming dense colonies under neomycin selection were transferred to BG-11 liquid medium for further cultivation. DNA samples from axenic cultures of the exconjugate strains were used in PCR screens for acsAB using primers which span a segment of acsA. As shown in Figure 2, a 2.2 Kb of PCR fragment corresponding to acsA was present in all four pPacsAB transgenic strains of Anabaena sp. PCC7120 (strain acsAB-2, strain acsAB-8, strain acsAB-17, and strain acsAB-26), but not in Anabae-na sp. PCC7120 wild type or Anabaena sp. PCC7120 cells harboring pRL489-Nm^r vector. The data indicated that the plasmid pPacsAB was successfully introduced into Ana-baena cells.

Determination of photosynthesis and respiration rate

Photosynthesis and respiration rate were determined by detecting the amount of O₂ evolution and consumption by algal cells using a Clark-type oxygen electrode fitted with a DW3 chamber (Hansatech, Kings Lynn, Norflok, England) thermostated at 28^oC. Ten mL of algal culture was centrifuged at 4,000 x g for 10 min at room temperature. The cells were resuspended in 10 mL of 50 mM HEPES-KOH buffer (pH 7.5) and transferred to a 20 mL square section reaction vessel containing 3 mM NaHCO3. The consumption of O2 as respiration was determined in the dark for 10 min and then exposed to 100 fmol photons m-2 s-1 for the determination of O2 evolution as net photosynthesis. The net photosynthesis rate and the respi-ration rate were expressed as the change in O2 concentra-tion per hour, and the gross photosynthetic rate was the value of 'net photosynthesis rate-respiration rate'. Three replicates per treatment were measured.

RT-PCR transcription analysis

In order to confirm the expression acsAB genes in trans-genic strains of Anabaena sp., RT-PCR-based transcription studies were performed. Four pPacsAB transgenic strains of Anabaena sp. strain PCC7120, that had grown well in BG-11 medium containing neomycin (Nm), strain acsAB-2, 8, 17 and 26, were used in RT-PCR analysis. Anabaena sp. strain PCC7120 wild type and Anabaena sp. strain PCC7120 cells harboring pRL489-Nm^r vector were used as negative controls. Total RNA was isolated from Anabaena PCC7120 wild type, and Anabaena PCC7120 cells harboring plasmid pRL489-Nm^r, pPacsAB grown under photoautotrophic conditions. cDNA produced in reverse transcription reaction was then used for PCR reactions with acsAB-F and acsA-R primers. As shown in Figure 3, a 2.2-kb band corresponding to acsA was detected

Chemicals and statistical analysis

Chemicals were purchased from Merck (Germany) or Sigma (USA). Statistics were analyzed by SAS (SAS v 9.01, NC, USA). Statistical differences between means (n = 3) were analyzed by Duncan's new multiple range test (P < 0.05) following significant analysis of variance (ANOVA, P < 0.05). ；

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in all four pPacsAB transgenic strains of Anabaena strain PCC7120, but not in Anabaena sp. strain PCC7120 wild type, or Anabaena sp. strain PCC7120 cells harboring pRL489-Nm^r vector. The results indicate that the acsAB genes are transcribed in the Anabaena sp. strain PCC7120 transgenic strains.

Determination of the location of cellulose by Calcofluor white staining and FITC-conjugated

CBHI

In order to detect the production of cellulose, Anabaena

PCC7120 wild type and Anabaena PCC7120 cells harboring plasmid pRL489-Nm^r, pPacsAB were analyzed by both Calcofluor white staining (Figure 4) and CBHI-FITC labeling (Figure 5). Calcofluor White Stain is a non-specific fluorescent brightening agent that binds to cellulose and chitin and is used extensively for the rapid detection of many yeasts and pathogenic fungi, and in analyzing the cellulosic content of Crypthecodinium cohnii cells (Kwok et al., 2007; Monheit et al., 1984). Cellobiohydrolase I (CBHI), from the fungus Trichoderma sp, is capable of binding to cellulose microfibrils or microcrystals (Chanzy et al., 1984) and has therefore been used to visualize the distribution of cellulose in a range of plant species and tissues (Berg et al., 1988; Benhamou, 1989; Berg, 1990; Bonfante-Fasolo et al., 1990; Roy and Vian, 1991; Fergu-son et al., 1998).

The results from Figures 4 and 5 show the extracellular matrix stained with Calcofluor white and FITC that was observed in Anabaena PCC7120 cells harboring plasmid pPacsAB. Little or no such extracellular matrix staining was observed in either wild type or Anabaena PCC7120 harboring plasmid pRL489-Nm^r. The presence of Calcofluor white and CBHI-FITC-stained extracellular material in cells harboring plasmid pPacsAB positively identifies cellulose as a component of the matrix. This is in agreement with previously published results (Nobles and Brown, 2008).

Figure 2. Colony PCR screen of Anabaena sp. strain PCC7120 exconjugates. From left to right: Lane M: marker; Lane 1 ： Negative control. (d?H₂O); Lane 2: pPacsAB plasmid; Lanes 3: Anabaena sp. PCC7120 wild type; Lane 4: Anabaena PCC7120 carrying plasmid pRL489-Nm^r; Lane 5-8: Anabaena PCC7120 carrying plasmid pPacsAB; Lane 5: strain acsAB-2; Lane 6: strain acsAB-8; Lane 7: strain acsAB-17; Lane 8: strain acsAB-26.

Glucose analysis

Calcofluor white stain is a non-specific fluorescent brightening agent that binds to cellulose and chitin. CBHI is comprised of a cellulose-binding domain linked to a catalytic domain by an extended O-glycosylated interdo-main peptide (Van Tilbeurgh et al., 1986; Srisodsuk et al.,1993; Reinikainen et al., 1995). CBHI is capable of bind-ing to cellulose as well as any other plant polysaccharide containing adjacent (1,4)-p-D-glucose residues, including mixed-link (1,3)-(1,4)- p-D-glucans from grasses (Bacic et al., 1988). Thus, in order to unambiguously determine the content of cellulose produced by these transgenic strains further cellulose hydrolysis was carried out.

Cellulose content was measured by determining the glucose levels in extracts of Anabaena PCC7120 wild type and Anabaena PCC7120 cells harboring either plasmid pRL489-Nm^r or pPacsAB with or without Celluclast hydrolysis. Glucose liberated from cellulose was determined

Figure 3. RT-PCR analysis of acsAB transcripts in Anabaena PCC7120 wild-type, and Anabaena PCC7120 cells carrying either pRL489-Nm^r or pPacsAB. From left to right: Lane M: marker; Lane 1: Negative control. (d?H₂O); Lane 2: pPacsAB plasmid; Lane 3: Anabaena PCC7120 wild-type; Lane 4: Ana-baena PCC7120 carrying plasmid pRL489-Nm^r; Lane 5-8: Anabaena PCC7120 carrying plasmid pPacsAB; Lane 5: strain acsAB-2; Lane 6: strain acsAB-8; Lane 7: strain acsAB-17; Lane 8: strain acsAB-26.

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by subtracting the concentration of glucose present in untreated extracts from that of extracts subjected to Cellu-clast digestion. As shown in Table 2, the glucose content of the acsAB transgenic strains acsAB-2 and acsAB-17 was significantly higher than that of either Anabaena PCC7120 wild type or a strain harboring pRL489-Nm^r. The data thus demonstrated that the pPacsAB transformants, strain acsAB-2 and acsAB-17, produce a significantly higher amount of cellulosic material than do the controls.

A limited number of studies have reported increased glucose production in transgenic cyanobacteria. One such study, carried out by Nobel and Brown (2008), involved the transfer of cellulose synthesis genes (acsABAC) from a heterotropic alpha proteobacterium to the cyanobacterium Synechococcus lepoliensis strain UTCC 100, resulting in a significant increase in the amount of cellulosic materials. Total glucose (mg ml^-1 OD₇₅₀^-1) increased from 0.08 to 0.26 (Nobles and Brown, 2008). The results obtained in this study demonstrated that Anabaena sp. strain PCC7120 wild type naturally produces much higher total glucose

than the Synechococcus lepoliensis strain UTCC 100 used in the Nobles study (0.20 vs 0.08 mg ml-1 OD750-1). After digestion with celluclast, our acsAB transgenic Anabaena sp. strain PCC7120 produced much higher levels of total glucose than the transgenic strain made by Nobles and Brown (0.53-0.66 vs 0.26 mg ml-1 OD750-1) (Table 2). The reasons for this difference remain to be determined.

Comparison of cell growth, chlorophyll a contents, photosynthesis and respiration

In order to examine whether enhanced cellulose synthesis impacts cell growth, the growth, respiration and photosynthesis rates were determined.

A comparison of OD and chlorophyll a content shows that the growth of the transformants harboring plasmid pPacsAB was significantly enhanced. After 15 days of incubation, all of the pPacsAB-containing transformants exhibited both higher OD values (Figure 6A) and chlorophyll a contents (Figure 6B) than those of the controls.

Table 2. Glucose assays.


		Glucose (mg ml^-1 D₇₅₀^-1)
Strains of PCC7120	Buffer	Celluclast digestion	Glucose from cellulose*
7120 wild type	0.041 ± 0.042	0.201 ± 0.017	0.160 ± 0.042
pRL489-Nm^r	0.057 ± 0.019	0.315 ± 0.053	0.258 ± 0.052
acsAB-2	0.103 ± 0.025	0.657 ± 0.227	0.553 ± 0.252
acsAB-17	0.093 ± 0.026	0.530 ± 0.097	0.436 ± 0.102

*Glucose liberated from cellulose was determined by subtracting the concentration of glucose present in Sodium Acetate buffer samples from the total glucose obtained from Celluclast digestions. Data are means ± SD (n = 3).

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Figure 5. Cellulose staining in different Anabaena strains using CBHI-FITC Probes. Bright field: A, C, E, G. Fluorescence: B, D, F, H. A-B: PCC7120 wild type. C-D: Anabaena sp. PCC7120 strains containing plasmid pRL489-Nm^r; E-F: strain acsAB-2; G-H: strain acsAB-26. Scale bars, 20 [im.

The net photosynthesis rate of all the transformants was twice as high than that of the wild type control (Figure 7). The respiration rate was significantly depressed in all transformants as compared to that of the controls (Figure 7). The gross photosynthesis rates of transfor-mants acsAB-2 and acsAB-17 were lower than those of the control, while the gross photosynthesis rates of the other transformants were the same as that of the wild type control (Figure 7).

The growth capacity and the overall photosynthesis capacity, based on O₂ evolution rate, were not negatively

affected in the acsAB transgenic Anabaena PCC7120 strains. In fact, the net photosynthesis was significantly increased in all the transformants. In contrast, the respiration rate in all tranformants exhibited a ~40% decline relative to the controls. It seems that the higher growth rate of the transformants may result from the down-regulation of respiration. We hypothesize that the overexpression of acsAB in Anabaena PCC7120 cells not only forces the redistribution of photosynthate for enhanced cellulose production but also reduces the wasteful respiratory utilization of photosynthate. Future studies will test this hypothesis.

SU et al. ― Enhanced cellulose production in Anabaena 273

Figure 6. The OD₇₅₀value (A) and chlorophyll a content (B) of Anabaena PCC7120 exconjugates and controls. Data are the means 士 SD (n = 3). Different symbol indicates significant differences at P < 0.05.

Figure 7. The gross photosynthesis (A), respiration (B) and net photosynthesis (C) rates of Anabaena PCC7120 exconjugates and controls. Data are the means ± SD (n = 3) and different symbol indicates significant difference at P < 0.05.

Acknowledgements. This work was supported by the Institute of Nuclear Energy Research, Atomic Energy Council, Executive Yuan, Taiwan, under grant number of 982001INER037. We are grateful to Dr. C. P. Wolk, Michigan State University, for kindly providing us plas-mids pRL489, pRL443, and pRL528; Dr. Mei-Chu Chung, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, for helpful discussions for the labeling of cel-lulose with FITC probes. Thank also to Academia Sinica, Taipei, Taiwan, for providing the fellowship of the Doctor Degree Program in Marine Biotechnology to Hsiang-Yen Su.

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275

絲狀藍綠菌表現異源纖維素合成酶基因促進纖維素合成、

光合作用與生長

蘇翔筵^1,4 李澤民^2,3,4 黃郁儒² 周聖圻⁵ 王嘉寶⁵ 林立夫⁵ 周德珍¹

¹輔英科技大學生物科技系

²國立中山大學海洋生物研究所

³國立中山大學亞太海洋研究中心黑潮研究群

⁴國立中山大學及中央研究院合辦海洋生物科技博士學位學程

⁵行政院原子能委員會核能研究所

微藻的纖維素與醣可用來生產生質酒精。而提高微藻的纖維素與醣的合成能力是以微藻大量生產
       生質酒精的關鍵。爲了增加纖維素產量'我們將Acetobacter xylinum纖維素合成酶基因acsAB以接合生
       殖方式轉入藍綠菌Anabaena sp. strain PCC7120細胞中。PCR分析結果確認轉殖株具有acsAB基因，而
       RT- PCR分析亦檢測出轉殖株中acsAB基因之表現。轉殖株培養液分別以Calcofluor white染色'及聯
       結FITC的纖維素水解酶CBHI-FITC爲探針檢測纖微素，皆發現acsAB轉殖株細胞外物質染色後出現螢
     光，顯示PCC7120 acsAB基因轉殖株分泌胞外纖維素。而與wild type相比'acsAB基因轉殖株經纖維
       素水解後的葡萄糖含量亦顯著的增加。轉殖株之光合作用效率分析結果顯示轉殖株的光合作用效率及生
        長速率提高。綜上所述'洳"b^enna sp. strain PCC7120表達acsAB不僅提高了纖維素的合成'對其光合
        作用及生長亦無負面影響。

關鍵詞：纖維素；纖維素合成酶；藍綠菌；光合作用。



	SU et al. — Enhanced cellulose production in Anabaena	275

	絲狀藍綠菌表現異源纖維素合成酶基因促進纖維素合成、光合作用與生長

	蘇翔筵^1,4 李澤民^2,3,4 黃郁儒² 周聖圻⁵ 王嘉寶⁵ 林立夫⁵ 周德珍¹

	¹輔英科技大學生物科技系 ²國立中山大學海洋生物研究所 ³國立中山大學亞太海洋研究中心黑潮研究群 ⁴國立中山大學及中央研究院合辦海洋生物科技博士學位學程 ⁵行政院原子能委員會核能研究所
	微藻的纖維素與醣可用來生產生質酒精。而提高微藻的纖維素與醣的合成能力是以微藻大量生產生質酒精的關鍵。爲了增加纖維素產量'我們將Acetobacter xylinum纖維素合成酶基因acsAB以接合生殖方式轉入藍綠菌Anabaena sp. strain PCC7120細胞中。PCR分析結果確認轉殖株具有acsAB基因，而 RT- PCR分析亦檢測出轉殖株中acsAB基因之表現。轉殖株培養液分別以Calcofluor white染色'及聯結FITC的纖維素水解酶CBHI-FITC爲探針檢測纖微素，皆發現acsAB轉殖株細胞外物質染色後出現螢光，顯示PCC7120 acsAB基因轉殖株分泌胞外纖維素。而與wild type相比'acsAB基因轉殖株經纖維素水解後的葡萄糖含量亦顯著的增加。轉殖株之光合作用效率分析結果顯示轉殖株的光合作用效率及生長速率提高。綜上所述'洳"b^enna sp. strain PCC7120表達acsAB不僅提高了纖維素的合成'對其光合作用及生長亦無負面影響。


	關鍵詞：纖維素；纖維素合成酶；藍綠菌；光合作用。